Method of modulating ribonucleotide reductase

ABSTRACT

A method of modulating ribonucleotide reductase activity in a neoplastic cell includes administering to the cell an amount of a ribonucleotide reductase allosteric modulator (RRAmod), the amount being effective to inhibit neoplastic cell growth.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/613,694, filed Mar. 21, 2012, the subject matter of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to selective modulators of ribonucleotidereductase (RR) and to methods of using such modulators for therapeuticapplications.

BACKGROUND

Ribonucleotide reductase (RR) is a highly regulated enzyme whichcatalyzes the de novo dNTP synthesis pathway that is ubiquitouslypresent in human, bacteria, yeast, and other organisms. RR plays acrucial role in de novo DNA synthesis by reducing ribonucleosidediphosphates to 2′-deoxy ribonucleoside diphosphates and maintainsbalanced pools of deoxynucleoside triphosphates (dNTPs) in the cell.

RRs are divided into three classes, I to III, based on the method offree-radical generation. All eukaryotic organisms encode a class I RR,consisting of an αnβn multi-subunit protein complex, in which theminimally active form is α2β2. The α or RR1 (large) subunit contains thecatalytic (C-site) and two allosteric sites, while the β or RR2 subunithouses a stable tyrosyl free radical that is transferred some 35 Å tothe catalytic site to initiate radical-based chemistry on the substrate.

RR is regulated transcriptionally, allosterically and, in the yeast S.cerevisiae, RR is further regulated by subunit localization and by itsprotein inhibitor Sm11. In mammalian cells, RR activity is alsocontrolled by the RR2 levels. Consistent with the varying RR2 levels,dNTP pools also vary with the phases of the cell cycle, reaching thehighest concentration during S-phase. RR is regulated by an intricateallosteric mechanism. The two allosteric sites of RR are the specificitysite (S-site), which determines substrate preference, and the activitysite (A-site), which stimulates or inhibits RR activity depending onwhether ATP or dATP is bound.

RR is directly involved in neoplastic tumor growth, metastasis, and drugresistance. The proliferation of cancer cells requires excess dNTPs forDNA synthesis. Therefore, an increase in RR activity is necessary as ithelps provide extra dNTPs for DNA replication in primary and metastaticcancer cells. Because of this critical role in DNA synthesis, RRrepresents an important target for cancer therapy. However, existingchemotherapies that target ribonucleotide reductase are nucleoside-basedanalogs. Hence they are promiscuous, leading to nonspecific binding ofother nucleoside binding proteins which results in unwanted sideeffects. Therefore, there is a need for compositions and methods forspecifically targeting and inhibiting RR activity in neoplastic cells inthe treatment of neoplastic disorders.

SUMMARY

Embodiments described herein relate to compounds and methods ofmodulating ribonucleotide reductase activity in a neoplastic cell. Themethod includes administering to the cell an amount of a ribonucleotidereductase allosteric modulator (RRAmod). The amount of RRAmod is theamount effective to inhibit neoplastic cell growth.

In another aspect, a method of treating a neoplastic disorder in asubject is provided. The method includes administering to neoplasticcells of the subject a therapeutically effective amount of apharmaceutical composition. The pharmaceutical composition includes anRRAmod. The therapeutically effective amount of an RRAmod is an amounteffective to inhibit neoplastic cell growth in the subject.

In yet another aspect, a pharmaceutical composition is provided. Thepharmaceutical composition includes an RRAmod. The RRAmod inhibits cellgrowth when administered to a neoplastic cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a ribbon diagram identifying the hexamer interface onribonucleotide reductase that is targeted by small molecules.

FIG. 2 is a graph showing the drug effects of Drug 1, Drug 2, Drug 3 andDrug 4 on the 231 triple negative breast cell line.

FIG. 3 is a graph showing the drug effects of Drug 1, Drug 2, Drug 3 andDrug 4 on the A549 non-small lung cell line.

FIG. 4 is a graph showing the drug effect of Drug 1 on the 231 triplenegative breast, A549 non-small lung and LN229 glioblastoma cell lines.

FIG. 5 is a graph showing the drug effect of Drug 2 on the 231 triplenegative breast, A549 non-small lung and LN229 glioblastoma cell lines.

FIG. 6 is a graph showing the drug effects of Drug 1, Drug 2, Drug 3 andDrug 4 on the LN229 glioblastoma cell line.

FIG. 7 is a graph showing the drug effect of Drug 3 (compound not shown)on the 231 triple negative breast, A549 non-small lung and LN229glioblastoma cell lines.

FIG. 8 is a graph showing the drug effect of Drug 4 on the 231 triplenegative breast, A549 non-small lung and LN229 glioblastoma cell lines.

FIG. 9 is a graph showing the drug effects of Drug 1, Drug 2, Drug 4 andGemcitabine on the Panc-1 pancreatic cell line.

FIG. 10 is a graph showing the drug effects of Drug 1, Drug 2, Drug 4and Gemcitabine on the HCT-116 colon cell line.

FIG. 11 is a graph showing the drug effects of Drug 1, Drug 2, Drug 4and Gemcitabine on the A549 non-small lung cell line.

FIGS. 12(A-B) are graphs illustrating the tryptophan fluorescencespectra of Hur1 (Human ribonucleotide reductase) and Hur1 in thepresence of Drug 4.

FIG. 13 illustrates a compound 2 increases cytotoxicity of gemcitabineof cis-platin. Growth inhibition studies on MDA-MB-231 human breastcarcinoma cell line in the presence of gemcitabine (F2CDP-red) orcis-platin (CDDP-blue) alone (solid lines), or in combination with anon-toxic does of compound 2 (dashed lines).

FIG. 14 illustrates that compound 3 is cytotoxic against human breastcarcinoma cells. Growth of the MDA-MB-231 cancer cell line is inhibitedby compound 3.

FIGS. 15(A-B) illustrate the structure of hRRM1 dimer with drug-targetsites mapped. The M-site is the hexamer interface, the A-site controlsactivity, the S-site controls specificity, the C-site is the catalyticsite, loop 1 and 2 mediate cross-talk between the S- and C-sites and theP-site binds the smaller R2 subunit derived peptide. (B)2|F_(o)|−|F_(c)| electron density (blue) of the phthalimide compoundcontoured at 1σ after refinement Lig plot analysis of compoundinteraction to hRRM1.

DETAILED DESCRIPTION

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise,” “comprising,” “include,” “including,” “have,” and“having” are used in the inclusive, open sense, meaning that additionalelements may be included. The terms “such as”, “e.g.”, as used hereinare non-limiting and are for illustrative purposes only. “Including” and“including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

The term “allosteric” refers to or denotes the alteration of theactivity of a protein (e.g., an enzyme) through the binding of aneffector molecule at a specific binding site. Effectors that decrease orincrease the protein's activity are referred to as “allostericmodulators”. An “allosteric site” as used herein relates to or denotesthe site on an enzyme molecule which binds with a nonsubstrate molecule,inducing a conformational change that results in an alteration of theaffinity of the enzyme for its substrate thereby modulating the enzyme'sactivity. The phrase “having the formula” or “having the structure” isnot intended to be limiting and is used in the same way that the term“comprising” is commonly used.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group typically although not necessarily containing 1 toabout 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well ascycloalkyl groups, such as cyclopentyl, cyclohexyl, and the like.Generally, although again not necessarily, alkyl groups herein contain 1to about 18 carbon atoms, preferably 1 to about 12 carbon atoms. Theterm “lower alkyl” intends an alkyl group of 1 to 6 carbon atoms.Substituents identified as “C₁-C₆ alkyl” or “lower alkyl” can contain 1to 3 carbon atoms, and more particularly such substituents can contain 1or 2 carbon atoms (i.e., methyl and ethyl). “Substituted alkyl” refersto alkyl substituted with one or more substituent groups, and the terms“heteroatom-containing alkyl” and “heteroalkyl” refer to alkyl in whichat least one carbon atom is replaced with a heteroatom, as described infurther detail infra. If not otherwise indicated, the terms “alkyl” and“lower alkyl” include linear, branched, cyclic, unsubstituted,substituted, and/or heteroatom-containing alkyl or lower alkyl,respectively.

The term “alkenyl” refers to a linear, branched or cyclic hydrocarbongroup of 2 to about 24 carbon atoms containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl,cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally,although again not necessarily, alkenyl groups can contain 2 to about 18carbon atoms, and more particularly 2 to 12 carbon atoms. The term“lower alkenyl” refers to an alkenyl group of 2 to 6 carbon atoms, andthe specific term “cycloalkenyl” intends a cyclic alkenyl group,preferably having 5 to 8 carbon atoms. The term “substituted alkenyl”refers to alkenyl substituted with one or more substituent groups, andthe terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer toalkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which atleast one carbon atom is replaced with a heteroatom. If not otherwiseindicated, the terms “alkenyl” and “lower alkenyl” include linear,branched, cyclic, unsubstituted, substituted, and/orheteroatom-containing alkenyl and lower alkenyl, respectively.

The term “alkynyl” refers to a linear or branched hydrocarbon group of 2to 24 carbon atoms containing at least one triple bond, such as ethynyl,n-propynyl, and the like. Generally, although again not necessarily,alkynyl groups can contain 2 to about 18 carbon atoms, and moreparticularly can contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6 carbon atoms. The term “substitutedalkynyl” refers to alkynyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkynyl” and“heteroalkynyl” refer to alkynyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkynyl” and “lower alkynyl” include linear, branched, unsubstituted,substituted, and/or heteroatom-containing alkynyl and lower alkynyl,respectively.

The term “alkoxy” refers to an alkyl group bound through a single,terminal ether linkage; that is, an “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. A “lower alkoxy” group intendsan alkoxy group containing 1 to 6 carbon atoms, and includes, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.Preferred substituents identified as “C₁-C₆ alkoxy” or “lower alkoxy”herein contain 1 to 3 carbon atoms, and particularly preferred suchsubstituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

The term “aryl” refers to an aromatic substituent containing a singlearomatic ring or multiple aromatic rings that are fused together,directly linked, or indirectly linked (such that the different aromaticrings are bound to a common group such as a methylene or ethylenemoiety). Aryl groups can contain 5 to 20 carbon atoms, and particularlypreferred aryl groups can contain 5 to 14 carbon atoms. Exemplary arylgroups contain one aromatic ring or two fused or linked aromatic rings,e.g., phenyl, naphthyl, biphenyl, diphenylether, diphenylamine,benzophenone, and the like. “Substituted aryl” refers to an aryl moietysubstituted with one or more substituent groups, and the terms“heteroatom-containing aryl” and “heteroaryl” refer to aryl, in which atleast one carbon atom is replaced with a heteroatom, as will bedescribed in further detail infra. If not otherwise indicated, the term“aryl” includes unsubstituted, substituted, and/or heteroatom-containingaromatic substituents.

The term “aryloxy” as used herein refers to an aryl group bound througha single, terminal ether linkage, wherein “aryl” is as defined above. An“aryloxy” group may be represented as —O-aryl where aryl is as definedabove. Preferred aryloxy groups contain 5 to 20 carbon atoms, andparticularly preferred aryloxy groups contain 5 to 14 carbon atoms.Examples of aryloxy groups include, without limitation, phenoxy,o-halo-phenoxy, m-halo-phenoxy, p-halo-phenoxy, o-methoxy-phenoxy,m-methoxy-phenoxy, p-methoxy-phenoxy, 2,4-dimethoxy-phenoxy,3,4,5-trimethoxy-phenoxy, and the like.

The term “alkaryl” refers to an aryl group with an alkyl substituent,and the term “aralkyl” refers to an alkyl group with an arylsubstituent, wherein “aryl” and “alkyl” are as defined above. Exemplaryaralkyl groups contain 6 to 24 carbon atoms, and particularly preferredaralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groupsinclude, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl,4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl,4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like.Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl,p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl,3-ethyl-cyclopenta-1,4-diene, and the like.

The term “cyclic” refers to alicyclic or aromatic substituents that mayor may not be substituted and/or heteroatom containing, and that may bemonocyclic, bicyclic, or polycyclic.

The terms “halo” and “halogen” are used in the conventional sense torefer to a chloro, bromo, fluoro or iodo substituent.

The term “heteroatom-containing” as in a “heteroatom-containing alkylgroup” (also termed a “heteroalkyl” group) or a “heteroatom-containingaryl group” (also termed a “heteroaryl” group) refers to a molecule,linkage or substituent in which one or more carbon atoms are replacedwith an atom other than carbon, e.g., nitrogen, oxygen, sulfur,phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly,the term “heteroalkyl” refers to an alkyl substituent that isheteroatom-containing, the term “heterocyclic” refers to a cyclicsubstituent that is heteroatom-containing, the terms “heteroaryl” andheteroaromatic” respectively refer to “aryl” and “aromatic” substituentsthat are heteroatom-containing, and the like. Examples of heteroalkylgroups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylatedamino alkyl, and the like. Examples of heteroaryl substituents includepyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl,imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples ofheteroatom-containing alicyclic groups are pyrrolidino, morpholino,piperazino, piperidino, etc.

The term “hydrocarbyl” refers to univalent hydrocarbyl radicalscontaining 1 to about 30 carbon atoms, preferably 1 to about 24 carbonatoms, more preferably 1 to about 18 carbon atoms, most preferably about1 to 12 carbon atoms, including linear, branched, cyclic, saturated, andunsaturated species, such as alkyl groups, alkenyl groups, aryl groups,and the like. “Substituted hydrocarbyl” refers to hydrocarbylsubstituted with one or more substituent groups, and the term“heteroatom-containing hydrocarbyl” refers to hydrocarbyl in which atleast one carbon atom is replaced with a heteroatom. Unless otherwiseindicated, the term “hydrocarbyl” is to be interpreted as includingsubstituted and/or heteroatom-containing hydrocarbyl moieties.

The terms “substituted” as in “substituted alkyl,” “substituted aryl,”and the like, as alluded to in some of the aforementioned definitions,is meant that in the alkyl, aryl, or other moiety, at least one hydrogenatom bound to a carbon (or other) atom is replaced with one or morenon-hydrogen substituents. Examples of such substituents include,without limitation: functional groups such as halo, hydroxyl, silyyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH₂),mono-(C₁-C₂₄ alkyl)-substituted carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),di-(C₁-C₄ alkyl)-substituted carbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂),mono-substituted arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl(—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN), isocyano (—N⁺C⁻),cyanato (—O—CN), isocyanato (—ON⁺C⁻), isothiocyanato (—S—CN), azido(—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), mono-and di-(C₁-C₂₄ alkyl)-substituted amino, mono- and di-(C₅-C₂₀aryl)-substituted amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C₁-C₂₄ alkyl,C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino(—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), and phosphino(—PH₂); and the hydrocarbyl moieties C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, and C₆-C₂₄ aralkyl.

In addition, the aforementioned functional groups may, if a particulargroup permits, be further substituted with one or more additionalfunctional groups or with one or more hydrocarbyl moieties such as thosespecifically enumerated above. Analogously, the above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl, alkenyl, andaryl” is to be interpreted as “substituted alkyl, substituted alkenyl,and substituted aryl.” Analogously, when the term“heteroatom-containing” appears prior to a list of possibleheteroatom-containing groups, it is intended that the term apply toevery member of that group. For example, the phrase“heteroatom-containing alkyl, alkenyl, and aryl” is to be interpreted as“heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the description includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

When referring to a compound described herein, the term “compound” ismeant to encompass not only the specified molecular entity but also itspharmaceutically acceptable, pharmacologically active analogs,including, but not limited to, salts, esters, amides, prodrugs,conjugates, active metabolites, and other such derivatives, analogs, andrelated compounds.

Some of the compounds disclosed herein may contain one or moreasymmetric centers and may thus give rise to enantiomers, diastereomers,and other stereoisomeric forms. The present invention is also meant toencompass racemic mixtures, resolved forms and mixtures thereof, as wellas the individual enantiomers that may be separated according to methodsthat are well know to those of ordinary skill in the art. When thecompounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended to include both E and Z geometric isomers.

The term “stereoisomers” is a general term for all isomers of individualmolecules that differ only in the orientation of their atoms in space.It includes enantiomers and isomers of compounds with more than onechiral center that are not mirror images of one another (diastereomers).

The term “asymmetric center” or “chiral center” refers to a carbon atomto which four different groups are attached.

The term “enantiomer” or “enantiomeric” refers to a molecule that isnonsuperimposeable on its mirror image and hence optically activewherein the enantiomer rotates the plane of polarized light in onedirection and its mirror image rotates the plane of polarized light inthe opposite direction.

The term “racemic” refers to a mixture of equal parts of enantiomers andwhich is optically inactive.

The term “resolution” refers to the separation or concentration ordepletion of one of the two enantiomeric forms of a molecule. The phrase“enantiomeric excess” refers to a mixture wherein one enantiomer ispresent is a greater concentration than its mirror image molecule.

The term “epitope” refers to a physical structure on a molecule thatinteracts with a selective component. In exemplary embodiments, epitoperefers to a desired region on a target molecule that specificallyinteracts with a selectivity component.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. When the term “pharmaceutically acceptable” isused to refer to a pharmaceutical carrier or excipient, it is impliedthat the carrier or excipient has met the required standards oftoxicological and manufacturing testing or that it is included on theInactive Ingredient Guide prepared by the U.S. Food and Drugadministration. “Pharmacologically active” (or simply “active”) as in a“pharmacologically active” derivative or analog, refers to a derivativeor analog having the same type of pharmacological activity as the parentcompound and approximately equivalent in degree.

The term “pharmaceutically acceptable salt” refers to a salt of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In some embodiments, the salt is an acidaddition salt of the compound. Pharmaceutical salts can be obtained byreacting a compound with inorganic acids such as hydrohalic acid (e.g.,hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid,phosphoric acid and the like. Pharmaceutical salts can also be obtainedby reacting a compound with an organic acid such as aliphatic oraromatic carboxylic or sulfonic acids, for example acetic, succinic,lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic,ethanesulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic acid.Pharmaceutical salts can also be obtained by reacting a compound with abase to form a salt such as an ammonium salt, an alkali metal salt, suchas a sodium or a potassium salt, an alkaline earth metal salt, such as acalcium or a magnesium salt, a salt of organic bases such asdicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine,C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, andsalts with amino acids such as arginine, lysine, and the like.

The term “small molecule” refers to a low molecular weight organiccompound, which is by definition not a polymer. The small molecule canbind with high affinity to a biopolymer, such as protein, nucleic acid,or polysaccharide and in some instances alter the activity or functionof the biopolymer. The upper molecular weight limit for a small moleculeis about 800 Daltons, which allows for the possibility to rapidlydiffuse across cell membranes so that they can reach intracellular sitesof action. In addition, this molecular weight cutoff is a necessary butinsufficient condition for oral bioavailability.

The term “anticancer agent” refers to a compound which treats a cancer(e.g., a compound which is useful in the treatment of a cancer). Theanticancer effect(s) may arise through one or more mechanisms including,but not limited to, the regulation of cell proliferation, the inhibitionof cell cycle progression, the inhibition of cell growth, the inhibitionof angiogenesis, the inhibition of metastasis, the inhibition ofinvasion (e.g., the spread of tumor cells into healthy neighboringtissue), or the promotion of apoptosis. The term “antineoplastic” isused herein to mean a chemotherapeutic intended to inhibit or preventthe maturation and proliferation of neoplasms, by targeting the DNA.

The term “cell growth” is used in the contexts of cell development andcell division (reproduction). When used in the context of cell division,it refers to growth of cell populations, where one cell (the “mothercell”) grows and divides to produce two “daughter cells” (M phase). Whenused in the context of cell development, the term refers to increase incytoplasmic and organelle volume (G1 phase), as well as increase ingenetic material before replication (G2 phase).

The terms “neoplastic cell”, “cancer cell” or “tumor cell” refer tocells that divide at an abnormal (i.e., increased) rate. A neoplasticcell or neoplasm (tumor) can be benign, potentially malignant, ormalignant. Cancer cells include, but are not limited to, carcinomas,such as squamous cell carcinoma, non-small cell carcinoma (e.g.,non-small cell lung carcinoma), small cell carcinoma (e.g., small celllung carcinoma), basal cell carcinoma, sweat gland carcinoma, sebaceousgland carcinoma, adenocarcinoma, papillary carcinoma, papillaryadenocarcinoma, cystadenocarcinoma, medullary carcinoma,undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cellcarcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma,cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma,choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas,gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma,prostate carcinoma, and squamous cell carcinoma of the neck and headregion; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, synoviosarcoma andmesotheliosarcoma; hematologic cancers, such as myelomas, leukemias(e.g., acute myelogenous leukemia, chronic lymphocytic leukemia,granulocytic leukemia, monocytic leukemia, lymphocytic leukemia),lymphomas (e.g., follicular lymphoma, mantle cell lymphoma, diffuselarge B-cell lymphoma, malignant lymphoma, plasmocytoma, reticulum cellsarcoma, or Hodgkin's disease), and tumors of the nervous systemincluding glioma, meningoma, medulloblastoma, schwannoma and epidymoma.

The term “subject” can be a vertebrate, such as a mammal, a fish, abird, a reptile, or an amphibian. Thus, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a mammal. A patient refers to a subjectafflicted with a disease or disorder (e.g., a neoplastic disorder). Theterm “patient” includes human and veterinary subjects. In some aspectsof the disclosed methods, the subject has been diagnosed with a need fortreatment of one or more neoplastic disorders prior to the administeringstep. In some aspects of the disclosed method, the subject has beendiagnosed with a need for allosteric inhibition of ribonucleotidereductase enzyme activity prior to the administering step.

The terms “treating” or “treatment” of a condition may refer topreventing or alleviating a condition, slowing the onset or rate ofdevelopment of a condition, reducing the risk of developing a condition,preventing or delaying the development of symptoms associated with acondition, reducing or ending symptoms associated with a condition,generating a complete or partial regression of a condition, curing acondition or some combination thereof. With regard to neoplasticdisorders, “treating” or “treatment” may refer to inhibiting or slowingneoplastic and/or malignant cell growth, proliferation, and/ormetastasis, preventing or delaying the development of neoplastic and/ormalignant cell growth, proliferation, and/or metastasis, or somecombination thereof. With regard to a tumor, “treating” or “treatment”may refer to eradicating all or part of a tumor, inhibiting or slowingtumor growth and metastasis, preventing or delaying the development of atumor, or some combination thereof.

The phrase “therapeutically effective amount” refers to an amount of acompound that produces a desired therapeutic effect. In one aspect, thetherapeutically effective amount is the amount required to inhibitneoplastic cell growth in the subject. The precise therapeuticallyeffective amount is an amount of the composition that will yield themost effective results in terms of efficacy in a given subject. Thisamount will vary depending upon a variety of factors, including but notlimited to the characteristics of the therapeutic compound (includingactivity, pharmacokinetics, pharmacodynamics, and bioavailability), thephysiological condition of the subject (including age, sex, disease typeand stage, general physical condition, responsiveness to a given dosage,and type of medication), the nature of the pharmaceutically acceptablecarrier or carriers in the formulation, and the route of administration.One skilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 22nd Edition, Pharmaceutical Press, London, UK, 2012).

The terms “IC₅₀,” or “half maximal inhibitory concentration” is intendedto refer to the concentration of a substance (e.g., a compound or adrug) that is required for 50% inhibition of a biological process, orcomponent of a process, including a protein, subunit, organelle,ribonucleoprotein, etc. In one aspect, an IC₅₀ can refer to theconcentration of a substance that is required for 50% inhibition of denovo DNA synthesis, cell growth or a surrogate thereof.

The terms “antibody” refers to an immunoglobulin, derivatives thereofwhich maintain specific binding ability, and proteins having a bindingdomain which is homologous or largely homologous to an immunoglobulinbinding domain. These proteins may be derived from natural sources, orpartly or wholly synthetically produced. An antibody may be monoclonalor polyclonal. The antibody may be a member of any immunoglobulin class,including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Inexemplary embodiments, antibodies used with the methods and compositionsdescribed herein are derivatives of the IgG class.

The term “antibody fragment” refers to any derivative of an antibodywhich is less than full-length. In exemplary embodiments, the antibodyfragment retains at least a significant portion of the full-lengthantibody's specific binding ability. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv, dsFvdiabody, and Fd fragments. The antibody fragment may be produced by anymeans. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody, it may berecombinantly produced from a gene encoding the partial antibodysequence, or it may be wholly or partially synthetically produced. Theantibody fragment may optionally be a single chain antibody fragment.Alternatively, the fragment may comprise multiple chains which arelinked together, for instance, by disulfide linkages. The fragment mayalso optionally be a multimolecular complex. A functional antibodyfragment will typically comprise at least about 50 amino acids and moretypically will comprise at least about 200 amino acids.

As used herein, the term “diabodies” refers to dimeric scFvs. Thecomponents of diabodies typically have shorter peptide linkers than mostscFvs and they show a preference for associating as dimers.

The term “epitope” refers to a physical structure on a molecule thatinteracts with a selective component. In exemplary embodiments, epitoperefers to a desired region on a target molecule that specificallyinteracts with a selectivity component.

The term “Fab” refers to an antibody fragment that is essentiallyequivalent to that obtained by digestion of immunoglobulin (typicallyIgG) with the enzyme papain. The heavy chain segment of the Fab fragmentis the Fd piece. Such fragments may be enzymatically or chemicallyproduced by fragmentation of an intact antibody, recombinantly producedfrom a gene encoding the partial antibody sequence, or it may be whollyor partially synthetically produced.

The term “Fab”′ refers to an antibody fragment that is essentiallyequivalent to that obtained by reduction of the disulfide bridge orbridges joining the two heavy chain pieces in the F(ab′)₂ fragment. Suchfragments may be enzymatically or chemically produced by fragmentationof an intact antibody, recombinantly produced from a gene encoding thepartial antibody sequence, or it may be wholly or partiallysynthetically produced.

The term “F(ab′)₂” refers to an antibody fragment that is essentiallyequivalent to a fragment obtained by digestion of an immunoglobulin(typically IgG) with the enzyme pepsin at pH 4.0-4.5. Such fragments maybe enzymatically or chemically produced by fragmentation of an intactantibody, recombinantly produced from a gene encoding the partialantibody sequence, or it may be wholly or partially syntheticallyproduced.

The term “Fv” refers to an antibody fragment that consists of one V_(H)and one V_(L) domain held together by noncovalent interactions. The term“dsFv” is used herein to refer to an Fv with an engineeredintermolecular disulfide bond to stabilize the V_(H)-V_(L) pair.

The terms “single-chain Fvs” and “scFvs” refers to recombinant antibodyfragments consisting of only the variable light chain (V_(L)) andvariable heavy chain (V_(H)) covalently connected to one another by apolypeptide linker. Either V_(L) or V_(H) may be the NH₂-terminaldomain. The polypeptide linker may be of variable length and compositionso long as the two variable domains are bridged without serious stericinterference. In exemplary embodiments, the linkers are comprisedprimarily of stretches of glycine and serine residues with some glutamicacid or lysine residues interspersed for solubility.

Embodiments described herein relate to ribonucleotide reductaseallosteric modulators (RRAmods), pharmaceutical compositions comprisingRRAmods, therapeutic uses of RRAmods, as well as compounds found to bespecifically effective as allosteric modulators of ribonucleotidereductase activity in neoplastic cells.

Using X-ray crystallography, we mapped a hexamer interface epitope ofthe large subunit of ribonuecleotide reductase (RR) (see FIG. 1) andfound that it could be targeted by small molecules to modulateribonucleotide reductase activity. The epitope found to be in thehexamer interface is part of an allosteric site of RR1, known as theactivity site (A-site). The epitope has the amino acid sequenceMHVIKRDGRQERVMFDKITSR (SEQ ID NO:1), which corresponds to residues 1 to21 of the 30 amino acid long N terminus of the RR1 subunit. Smallmolecules that bind to or complex with this region of ribonucleotidereductase were found to allosterically modulate (e.g., inhibit oractivate) the enzyme.

Ribonucleotide reductase enzyme activity is required for de novo DNAsynthesis by catalyzing ribonucleotides to deoxy ribonucleotides andmaintaining a balanced nucleotide precursor molecule pool. Since theproliferation of cancer cells requires excess dNTPs for DNA synthesis,RRAmods that specifically target the hexamer interface of RR1 can beemployed to inhibit cell growth and proliferation of neoplastic cellsthrough the modulation of ribonucleotide reductase enzyme activity.Therefore, in some embodiments described herein, a method of modulatingribonucleotide reductase activity in a neoplastic cell can includeadministering to the neoplastic cell an amount of an RRAmod effective toinhibit neoplastic cell growth.

RRAmods described herein include agents capable of binding to orcomplexing with the hexamer interface of ribonucleotide reductase andallosterically modulating ribonucleotide reductase enzyme activity,thereby affecting de novo DNA synthesis, cell growth and proliferationof neoplastic cells. RRAmods can include organic compounds, peptides,inorganic compounds, lipids, peptidomimetics, antibodies or fragmentsthereof and small molecule synthetic compounds.

In some embodiments, the RRAmod is an antibody that selectively orspecifically binds to the hexamer interface of ribonucleotide reductase.In certain embodiments, the antibody can specifically bind to SEQ IDNO: 1. The antibody can be a monoclonal antibody, a polyclonal antibody,or a humanized antibody including without limitation: Fv fragments,single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2 fragments,single domain antibodies, camelized antibodies and antibody fragments,humanized antibodies and antibody fragments, and multivalent versions ofthe foregoing; multivalent targeting moieties including withoutlimitation: monospecific or bispecific antibodies, such as disulfidestabilized Fv fragments, scFv tandems ((scFv)₂ fragments), diabodies,tribodies or tetrabodies, which typically are covalently linked orotherwise stabilized (i.e., leucine zipper or helix stabilized) scFvfragments; and receptor molecules, which naturally interact with adesired target molecule.

Preparation of antibodies may be accomplished by any number ofwell-known methods for generating monoclonal antibodies. These methodstypically include the step of immunization of animals, typically mice,with a desired immunogen (e.g., a desired target molecule or fragmentthereof). Once the mice have been immunized, and preferably boosted oneor more times with the desired immunogen(s), monoclonalantibody-producing hybridomas may be prepared and screened according towell known methods. See, for example, Kuby, Janis, Immunology, ThirdEdition, pp. 131-139, W.H. Freeman & Co. (1997), for a general overviewof monoclonal antibody production, that portion of which is incorporatedherein by reference.

In certain embodiments, the RRAmod is a small molecule. Exemplary dataof small molecule compounds found to be specifically effective asallosteric modulators of ribonucleotide reductase activity are providedin the Example below. In particular, the disclosed compounds hadactivity in modulating the ribonucleotide reductase activity in DNAsynthesis assays, generally with an IC₅₀ for inhibition or activation ofless than about 70 μM. Certain compounds described herein have an IC₅₀for killing carcinomas in a cell-based assay of less than about 2 μM.

In one embodiment, an RRAmod can include a small molecule having theformula (I):

wherein each of R₁ to R₂₀ can be independently selected from the groupconsisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), imino (—CR═NH whereR is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations thereof, andwherein adjacent R groups may be linked to form a cyclic or polycyclicring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, orheterocyclic; or a pharmaceutically acceptable salt thereof.

In other embodiments, each of R₁ to R₂₀ can be independently selectedfrom hydrogen, alkyl, branched alkyl, cyclo-alkyl, halogen, a heteratom,or a pharmaceutically acceptable salt thereof.

In certain embodiments, an RRAmod having formula (I) can be:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the RRAmod can be a small molecule having theformula (II):

wherein each of R₅₄ to R₆₂ can be independently selected from the groupconsisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), imino (—CR═NH whereR is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations thereof, andwherein adjacent R groups may be linked to form a cyclic or polycyclicring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, orheterocyclic; or a pharmaceutically acceptable salt thereof.

In certain embodiments, an RRAmod having formula (II) can have thefollowing formula (III):

wherein each of R₅₄ to R₆₀ can be independently selected from the groupconsisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), imino (—CR═NH whereR is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations thereof, andwherein adjacent R groups may be linked to form a cyclic or polycyclicring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, orheterocyclic; or a pharmaceutically acceptable salt thereof.

In some embodiments, an RRmodA having formula (III) can be selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof.

In still other embodiments, an RRAmod having formula (II) can have thefollowing formula:

or be a pharmaceutically acceptable salt thereof.

In another embodiment, the RRAmod can be a small molecule having theformula (IV):

wherein each of R₄₆ to R₅₃ can be independently selected from the groupconsisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), imino (—CR═NH whereR is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations thereof, andwherein adjacent R groups may be linked to form a cyclic or polycyclicring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, orheterocyclic; or a pharmaceutically acceptable salt thereof.

In certain embodiments, an RRAmod having formula (IV) can have thefollowing formula (V):

wherein R₄₆ can be selected from the group consisting of hydrogen,C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, C₆-C₂₄alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl(including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀ arylcarbonyl(—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl),C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato(—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl), carboxy(—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), imino (—CR═NH whereR is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations thereof, ora pharmaceutically acceptable salt thereof.

In some embodiments, an RRmodA having formula (V) can be selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof.

In certain embodiments, an RRAmod having formula (IV) can have thefollowing formula (VI):

wherein R₄₆ can be selected from the group consisting of hydrogen,C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, C₆-C₂₄alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl(including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀ arylcarbonyl(—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl),C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato(—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl), carboxy(—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), imino (—CR═NH whereR is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations thereof, ora pharmaceutically acceptable salt thereof.

In some embodiments, an RRmodA having formula (VI) can be:

or a pharmaceutically acceptable salt thereof.

In another embodiment, an RRAmod can include a small molecule having theformula (VII):

wherein X₁ is a C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, Y₁ is aC₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, and R₄₅ is hydrogen, aC₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, an amino (—NH₂), or—(C₁-C₂₄ alkyl)-amino, or a pharmaceutically acceptable salt thereof.

In certain embodiments, an RRAmod having formula (VII) can be selectedfrom the group consisting of:

and a pharmaceutically acceptable salt thereof.

In another embodiment, an RRAmod can include a small molecule having theformula (VIII).

wherein R₆₃ can be selected from the group consisting of hydrogen,C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, C₆-C₂₄alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl(including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C⁶-C₂₀ arylcarbonyl(—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl),C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato(—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl), carboxy(—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano(—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), imino (—CR═NH whereR is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), combinations thereof, ora pharmaceutically acceptable salt thereof.

In certain embodiments, an RRAmod having formula (VIII) can be:

or a pharmaceutically acceptable salt thereof.

In another embodiment, an RRAmod can include a small molecule having theformula (IX):

wherein each R₄₁ to R₄₅ can be independently selected from hydrogen,alkyl, branched alkyl, cyclo-alkyl, halogen, a heteroatom, or apharmaceutically acceptable salt thereof.

In certain embodiments, an RRAmod having formula (VIII) can be:

or a pharmaceutically acceptable salt thereof.

In still other embodiments, the RRAmod can be a small molecule selectedfrom the group consisting of:

and pharmaceutically acceptable salts thereof.

Additional RRAmods can be identified by screening compounds for theability to modulate (e.g., inhibit or activate) ribonucleotide reductaseenzyme activity. Candidate RRAmods can be screened for function by avariety of techniques known in the art and/or disclosed within theinstant application. Candidate compounds may be screened individually,in combination, or as a library of compounds.

Examples of additional RRAmods identified as having the ability toinhibit or activate ribonucleotide reductase enzyme activity include:

or pharmaceutically acceptable salts thereof.

Candidate compounds screened include chemical compounds. In someaspects, the candidate compound is a small organic molecule having amolecular weight of more than about 50 and less than about 2,500daltons. Compounds screened are also found among biomolecules including,but not limited to: peptides, saccharides, fatty acids, steroids,pheromones, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. The compounds screened can include functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl, or carboxyl group.

Candidate compounds can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. Compounds to bescreened can be produced, for example, by bacteria, yeast or otherorganisms (e.g., natural products), produced chemically (e.g., smallmolecules, including peptidomimetics), or produced recombinantly. It isfurther contemplated that natural or synthetically produced librariesand compounds are readily modified through conventional chemical,physical and biochemical means, and may be used to produce combinatoriallibraries. Known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs.

In many drug screening programs, with test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays of described herein may be developed with purified orsemi-purified proteins or with lysates. These assays are often preferredas “primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target, which is mediated by a test agent. Assays describedherein can include cell-based assays. Cell-based assays may be performedas either a primary screen, or as a secondary screen to confirm theactivity of compounds identified in a cell free screen, such as an insilico screen.

Embodiments described herein also relate to a method of screening insilico for a compound effective as an RRAmod. For example, a 3-D modelof the hexamer interface epitope of RR1 targeted by small molecules canbe used to provide a pharmacophore using X-ray Crystallography. Aninitial model can then be generated using a suitable protein modelingsoftware program. In some aspects, the model can then be subjected toenergy refinement with a software program such as SURFLEX dock. Thepharmacophore can be modified to comply to the Lipinski limits to designdrug-like molecules with good bioavailability. In one embodiment, thetemplate used for docking was the hexamer interface of ribonucleotidereductase as shown in FIG. 1.

Once a model is built, small molecule RRAmods that bind toribonucleotide reductase at the hexamer interface of RR1 can beidentified by methods well known in the relevant art using in silicoconformation screening techniques. For example, virtual screening of theUniversity of Cincinnati Drug Discovery Center (UC DCC) Library of350,000 compounds can be performed using the drug discovery softwareSYBYLX1.3 (Tripos, St. Louis, Mo.). Such software can also be used todesign modified analogs of compounds for use as RRAmods. In parallel,ZINC and other commercial databases can be searched using withinSYBYLX1.3 software for lead compounds that satisfy the pharmacophore.These hits can be docked and scored using SURFLEX dock option inSYBYLX1.3. The best hits can then be discriminated using two scoringfunctions called, a docking score and the C-score. The docking score istheoretically equivalent to the negative logarithm of K_(d), whileC-score is a consensus scoring function. Hence, docking scores that areequal to 6 would mean a theoretical K_(d) of micromolar. The maximumC-score that can be obtained is five. Based on these criteria, aftervirtually screening the library, the best scoring candidates can beselected and then tested using various in vitro and cell based assaysdescribed herein and known in the art for efficacy. The larger numbersobtained for dock score and C-scores greater than 6 and 4-5 respectivelyrepresents the high ranking inhibitors that are predicted to have highaffinities.

In some aspects, about 20,000 compounds can be selected from in silicoscreening for an in vitro high-throughput screening (HTS). HTS can becarried out using an automated HTS system which performs biochemical andcell-based assays using 96 or 384-well microtiter plates. The systemincludes detectors, CO₂ incubators, pipetting systems, a plate washer,centrifuge, a storage unit, bar code readers, xyz robots, turntables,and pushers necessary for fully automated screening. A Jobin Yvon-Spexfluorescence spectrophotometer can be used to record the spectra.Alternatively, a multimode PERKIN-ELMER plate reader can be used fordetecting fluorescence intensity, fluorescence polarization,fluorescence resonance energy transfer, luminescence, or absorbanceusing ZEISS optics and a sensitive CCD camera. The PERKIN-ELMER Operadetector performs high content screening using confocal microscopy andimage analysis software powered by onboard servers. Lasers and CCDcameras allow measurement of subcellular localization, binding events orany other microscopic images which can be rapidly quantitated. Imageanalysis is performed immediately after the image is captured and storedin a database. All other data can be analyzed using GENEDATA HTSanalysis software (Switzerland), stored in a GENEDATA database based onORACLE.

In some embodiments, in vitro HTS includes a fluorescence based assayadapted for HTS. For example, in vitro HTS can employ tryptophanfluorescence quenching. The binding sites of proteins are known to oftencontain tryptophan (Trp) residues, whose fluorescent properties may bealtered upon ligand binding. Conformational changes within the bindingsite or simply the presence of the ligand can result in eitherfluorescence quenching or enhancement, which may be utilized toquantitatively investigate protein-ligand interactions. Change inintrinsic tryptophan fluorescence is used to measure the binding of acandidate agent to the Sml1 binding site of ribonucleotide reductase. Asshown in the Example below and in FIG. 12, the trytophan fluorescencespectra of Hur1 (Human ribonucleotide reductase) and a candidatecompound can be recorded and then compared in order to determine theextent of quenching. The ribonucleotide reductase samples are titratedwith 65 μM candidate compounds at room temperature where a decrease influorescence, or quenching, can be correlated with the binding affinityof the candidate compound to the Sml1 binding site of ribonucleotidereductase and/or a conformational change in the ribonucleotide reductaseSml1 binding site.

In some aspects, candidate RRAmod compounds, including those collectedfrom an in silico similarity search or HTS assay, may be furtherscreened for efficacy using in vitro and/or in vivo experimentalscreening methods known in the art. The efficacy of an identifiedcompound can be assessed by generating dose response curves from dataobtained using various concentrations of the test compound. Moreover, acontrol assay can also be performed to provide a baseline forcomparison. Such candidates can be further tested for their effects oncancer and tumor cell growth, proliferation, apoptosis, differentiation,and transformation properties compared to controls as well as theirability to: inhibit de novo DNA synthesis in vitro; unbalance nucleotidepool of DNA precursor molecules in vitro; modulate ribonucleotidereductase activity in vitro; and/or for other properties, such as theability to inhibit cell growth and increase the toxicity of neoplasticcells in vivo.

In some embodiments, assays used for in vitro screening of candidatecompounds for cell growth inhibition can include DNA synthesis assaysand MTT colorimetric assays to measure cell metabolism. For example, aDNA synthesis assay can include the steps of: (a) contacting theneoplastic cell with various concentrations of a candidate compound; and(b) comparing the DNA synthesis of the cell in step (a) with the DNAsynthesis of the cell in the absence of the compound so as to determinewhether the compound significantly inhibits ribonucleotide reductaseactivity, thereby reducing the growth of the cell. One can alsodetermine the IC₅₀ of a candidate compound if the compound is found tosignificantly inhibit ribonucleotide reductase activity. The IC₅₀ of adrug can be determined by constructing a

dose-response curve and examining the effect of different concentrationsof a candidate agent on cell growth and/or ribonucleotide reductaseenzyme activity. IC₅₀ values can be calculated for a given compound bydetermining the concentration needed to inhibit half of the maximumbiological response of the compound.

For in vivo screening of candidate compounds, the candidate compound canbe administered in any manner desired and/or appropriate for delivery ofthe compound in order to affect a desired result. For example, thecandidate compound can be administered to a mammalian subject byinjection (e.g., by injection intravenously, intramuscularly,subcutaneously, or directly into the tissue in which the desired affectis to be achieved), topically, orally, or by any other desirable means.

Normally, this screen will involve a number of animals receiving varyingamounts and concentrations of the candidate compounds (from no compoundto an amount of compound that approaches an upper limit of the amountthat can be delivered successfully to the animal), and may includedelivery of the compound in different formulations. The compounds can beadministered singly or can be combined in combinations of two or more,especially where administration of a combination of compounds may resultin a synergistic effect.

The effect of compound administration upon the animal model can bemonitored by any suitable method such as assessing the number and sizeof tumors, overall health, survival rate, etc. A candidate compound isidentified as an effective compound for use in the treatment of aneoplastic disorder in a subject where candidate compound inhibitsneoplastic cell growth in the animal in a desirable manner (e.g., bybinding to the Sml1 allosteric binding site of ribonucleotide reductaseand allosterically inhibiting the enzyme's activity, etc.). In someaspects, effective compounds can be identified as having low toxicity invivo.

As shown in the Examples below, RRAmods disclosed herein have been shownto bind to the hexamer interface of RR1 and inhibit growth of multiplecancer cell types in vitro, supporting the use of these RRAmods to treata wide range of neoplastic diseases and disorders. Thus, in accordancewith another embodiments, RRAmods described herein can be used for thepreparation of a pharmaceutical composition for the treatment of aneoplastic disorder in a subject. In one embodiment, the subject issuffering from a neoplastic disorder characterized by increased cellgrowth. In another embodiment, the subject is suffering from cancer.

A therapeutically effective amount of a RRAmod described herein can beadministered to a subject for the treatment of a variety of conditionsin order to inhibit cell growth in the subject. Such conditions include,without being limited thereto, neoplastic disorder, and in particularall types of solid tumors; skin proliferative diseases (e.g.,psoriasis); and a variety of benign hyperplasic disorders.

In one aspect, the neoplastic disorder is cancer. The cancer caninclude, but is not limited to, carcinomas, such as squamous cellcarcinoma, non-small cell carcinoma (e.g., non-small cell lungcarcinoma), small cell carcinoma (e.g., small cell lung carcinoma),basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma,adenocarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma,bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-livercell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillarycarcinoma, transitional cell carcinoma, choriocarcinoma, semonoma,embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma,colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamouscell carcinoma of the neck and head region; sarcomas, such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; hematologiccancers, such as myelomas, leukemias (e.g., acute myelogenous leukemia,chronic lymphocytic leukemia, granulocytic leukemia, monocytic leukemia,lymphocytic leukemia), lymphomas (e.g., follicular lymphoma, mantle celllymphoma, diffuse large B-cell lymphoma, malignant lymphoma,plasmocytoma, reticulum cell sarcoma, or Hodgkin's disease), and tumorsof the nervous system including glioma, meningoma, medulloblastoma,schwannoma and epidymoma. In certain aspects, the cancer is apancreatic, breast, lung, colon or glyoblastoma cancer.

In another aspect, the neoplastic disorder is a solid tumor. Exemplarysolid tumors include carcinomas, sarcomas, adenomas, and cancers ofneuronal origin and if fact to any type of cancer which does notoriginate from the hematopoeitic cells and in particular concerns:carcinoma, sarcoma, adenoma, hepatocellular carcinoma,hepatocellularcarcinoma, hepatoblastoma, rhabdomyosarcoma, esophagealcarcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma,myxosarcoma, liposarcoma, cohndrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphagiosarcoma, synovioama, Ewing'stumor, leimyosarcoma, rhabdotheliosarcoma, colon carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma,hematoma, bile duct carcinoma, melanoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor,lung carcinoma, small lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma,ependynoma, pinealoma, retinoblastoma, multiple myeloma, rectalcarcinoma, thyroid cancer, head and neck cancer, brain cancer, cancer ofthe peripheral nervous system, cancer of the central nervous system,neuroblastoma, cancer of the endometrium, as well as metastasis of allthe above.

Benign hyperplasic disorders include, without being limited thereto,benign prostate hyperplasia (BPH), non-tumorigenic polyps in thedigestive tract, in the uterus and others.

In addition to cancer, the RRAmods disclosed herein may be used to treatother conditions associated with aberrant ribonucleotide reductaseenzyme activity such as for example various mitochondrial,redox-related, degenerative diseases, and viruses such as HIV.

When used as therapeutic agents in the treatment of neoplasticdisorders, the RRAmods can be conveniently formulated intopharmaceutical formulations composed of one or more of the compounds(e.g., RRAmods of formulas (I-III) or an RRAmodidentified by a screeningassay as described above) in association with a pharmaceuticallyacceptable carrier or excipient. (See Remington: The Science andPractice of Pharmacy (Gennaro ed. 22nd Edition, Pharmaceutical Press,London, UK, 2012), which discloses typical carriers and conventionalmethods of preparing pharmaceutical formulations).

In making the compositions, the RRAmod is usually mixed with theexcipient, diluted by an excipient or enclosed within a carrier whichcan be in the form of a capsule, sachet, paper or other container. Whenthe excipient serves as a diluent, it can be a solid, semi-solid, orliquid material, which acts as a vehicle, carrier or medium for theRRAmod. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium), soft andhard gelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders. The RRAmods can also be administered to asubject as a stabilized prodrug to increase the activity,bioavailability, stability or otherwise alter the properties of theRRAmod.

The effective amount of RRAmod in the pharmaceutical composition andunit dosage form thereof may be varied or adjusted widely depending uponthe particular application, the manner or introduction, the potency ofthe particular compound, and the desired concentration.

The effective amount is typically determined in appropriately designedclinical trials (dose range studies) and the person versed in the artwill know how to properly conduct such trials in order to determine theeffective amount. As generally known, an effective amount depends on avariety of factors including the affinity of the RRAmod to theallosteric Sml1 binding site, its distribution profile within the body,a variety of pharmacological parameters such as half life in the body,on undesired side effects, if any, on factors such as age and gender,etc.

The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient.

In this case, the composition will typically be administered over anextended period of time in a single daily dose, in several doses a day,as a single dose and in several days, etc. The treatment period willgenerally have a length proportional to the length of the diseaseprocess and the specific RRAmod effectiveness and the patient speciesbeing treated.

RRAmods and pharmaceutical compositions thereof can be administered tothe subject by any suitable means, including, for example, oral,intravenous, intramuscular, intra-arterial, subcutaneous, intranasal,via the lungs (inhalation) and through local administration.

RRAmods described herein can be used as single agents or in combinationor in conjunction with one or more other therapeutic agents in thetreatment of the aforementioned diseases, disorders and conditions forwhich RRAmods or the other agents have utility. In some embodiments, acombination of an RRAmod and other therapeutic agent together is saferor more effective than either drug alone.

In some embodiments, the other therapeutic agent used in a combinationtherapy can include at least one anti-proliferative agent selected fromthe group consisting at least one of a chemotherapeutic agent, ananticancer agent, an antimetabolite, a DNA damaging agent, anantitumorgenic agent, an antimitotic agent, an antiviral agent, anantineoplastic agent, an immunotherapeutic agent, and a radiotherapeuticagent. Additional therapeutic agents used in combination therapies withRRAmods can include biguanides (e.g., metformin, phenformin andbuformin), AP endonuclease inhibitors (e.g., methoxyamine (MX)), BERinhibitors including PARP inhibitors, and ribonucleotide reductaseinhibiting agents. Exemplary ribonucleotide reductase inhibiting agentsfor use in conjunction with RRAmods include O⁶-methyl-arabinofuranosylguanine (nelarabine), 2′-fluro-2′-deoxyarabinofuranosyl-2-chloroadenine(clofarabine), N⁴-pentyloxycarbonyl-5′-deoxy-5-flurocytidine(capecitabine), 2,2-difluoro-2′-deoxyadenosine (cladribine),arabinofuranosyl-2-fluoroadenine (fludarabine), 2′-deoxycoformycin(pentostatin), 5-fluro-2′deoxyuridine, arabinofuranosylcytosine(cytarabine), 6-thioguanine, 5-fluorouracil, methotrexate,6-mercaptopurine.

In some aspects, RRAmods can be used in a combination therapy with ananti-proliferative agent. The phrase “anti-proliferative agent” caninclude agents that exert antineoplastic, chemotherapeutic, antiviral,antimitotic, antitumorgenic, and/or immunotherapeutic effects, e.g.,prevent the development, maturation, or spread of neoplastic cells,directly on the tumor cell, e.g., by cytostatic or cytocidal effects,and not indirectly through mechanisms such as biological responsemodification. There are large numbers of anti-proliferative agent agentsavailable in commercial use, in clinical evaluation and in pre-clinicaldevelopment, which can be included by combination drug chemotherapy. Forconvenience of discussion, anti-proliferative agents are classified intothe following classes, subtypes and species: ACE inhibitors, alkylatingagents, angiogenesis inhibitors, angiostatin, anthracyclines/DNAintercalators, anti-cancer antibiotics or antibiotic-type agents,antimetabolites, antimetastatic compounds, asparaginases,bisphosphonates, cGMP phosphodiesterase inhibitors, calcium carbonate,cyclooxygenase-2 inhibitors, DHA derivatives, DNA topoisomerase,endostatin, epipodophylotoxins, genistein, hormonal anticancer agents,hydrophilic bile acids (URSO), immunomodulators or immunological agents,integrin antagonists, interferon antagonists or agents, MMP inhibitors,miscellaneous antineoplastic agents, monoclonal antibodies,nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATTs,radio/chemo sensitizers/protectors, retinoids, selective inhibitors ofproliferation and migration of endotheliai cells, selenium, stromelysininhibitors, taxanes, vaccines, and vinca alkaloids.

The major categories that some anti-proliferative agents fall intoinclude antimetabolite agents, alkylating agents, antibiotic-typeagents, hormonal anticancer agents, immunological agents,interferon-type agents, and a category of miscellaneous antineoplasticagents. Some anti-proliferative agents operate through multiple orunknown mechanisms and can thus be classified into more than onecategory.

A first family of anti-proliferative agents, which may be used incombination therapy with an RRAmod consists of antimetabolite-typeanti-proliferative agents. Antimetabolites are typically reversible orirreversible enzyme inhibitors, or compounds that otherwise interferewith the replication, translation or transcription of nucleic acids.Examples of antimetabolite antineoplastic agents that may be usedinclude, but are not limited to acanthifolic acid, aminothiadiazole,anastrozole, bicalutamide, brequinar sodium, capecitabine, carmofur,Ciba-Geigy CGP-30694, cladribine, cyclopentyl cytosine, cytarabinephosphate stearate, cytarabine conjugates, cytarabine ocfosfate, LillyDATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine,didox, Yoshitomi DMDC, doxifluridine, Wellcome EHNA, Merck & Co. EX-015,fazarabine, finasteride, floxuridine, fludarabine phosphate,N-(2′-furanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, fluorouracil(5-FU), 5-FU-fibrinogen, gemcitabine, isopropyl pyrrolizine, LillyLY-188011, Lilly LY-264618, methobenzaprim, methotrexate, WellcomeMZPES, nafarelin, norspermidine, nolvadex, NCI NSC-127716, NCINSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA,pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, stearate;Takeda TAC-788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate,tyrosine kinase inhibitors, tyrosine protein kinase inhibitors, TaihoUFT, toremifene, and uricytin, all of which are disclosed in U.S. Pat.No. 6,916,800, which is herein incorporated by reference in itsentirety.

A second family of anti-proliferative agents, which may be used incombination therapy with the RRAmods, consists of alkylating-typeanti-proliferative agents. The alkylating agents are believed to act byalkylating and cross-linking guanine and possibly other bases in DNA,arresting cell division. Typical alkylating agents include nitrogenmustards, ethyleneimine compounds, alkyl sulfates, cisplatin, andvarious nitrosoureas. A disadvantage with these compounds is that theynot only attack malignant cells, but also other cells which arenaturally dividing, such as those of bone marrow, skin,gastro-intestinal mucosa, and fetal tissue. Examples of alkylating-typeanti-proliferative agents that may be used include, but are not limitedto, Shionogi 254-S, aldo-phosphamide analogues, altretamine, anaxirone,Boehringer Mannheim BBR-2207, bestrabucil, budotitane, Wakunaga CA-102,carboplatin, carmustine (BiCNU), Chinoin-139, Chinoin-153, chlorambucil,cisplatin, cyclophosphamide, American Cyanamid CL-286558, Sanofi CY-233,cyplatate, dacarbazine, Degussa D-19-384, Sumimoto DACHP(Myr)2,diphenylspiromustine, diplatinum cytostatic, Erba distamycinderivatives, Chugai DWA-2114R, ITI E09, elmustine, Erbamont FCE-24517,estramustine phosphate sodium, etoposide phosphate, fotemustine, UnimedG-6-M, Chinoin GYKI-17230, hepsul-fam, ifosfamide, iproplatin,lomustine, mafosfamide, mitolactol, mycophenolate, Nippon Kayaku NK-121,NCI NSC-264395, NCI NSC-342215, oxaliplatin, Upjohn PCNU, prednimustine,Proter PTT-119, ranimustine, semustine, SmithKline SK&F-101772,thiotepa, Yakult Honsha SN-22, spiromus-tine, Tanabe Seiyaku TA-077,tauromustine, temozolomide (TMZ), teroxirone, tetraplatin andtrimelamol.

A third family of anti-proliferative agents that may be used incombination therapy with the RRAmods consists of antibiotic-typeanti-proliferative agents. Examples of antibiotic-typeanti-proliferative agents that may be used include, but are not limitedto Taiho 4181-A, aclarubicin, actinomycin D, actinoplanone, ErbamontADR-456, aeroplysinin derivative, Ajinomoto AN-201-II, Ajinomoto AN-3,Nippon Soda anisomycins, anthracycline, azino-mycin-A, bisucaberin,Bristol-Myers BL-6859, Bristol-Myers BMY-25067, Bristol-Myers BMY-25551,Bristol-Myers BMY-26605, Bristol-Myers BMY-27557, Bristol-MyersBMY-28438, bleomycin sulfate, bryostatin-1, Taiho C-1027, calichemycin,chromoximycin, dactinomycin, daunorubicin, Kyowa Hakko DC-102, KyowaHakko DC-79, Kyowa Hakko DC-88A, Kyowa Hakko DC89-A1, Kyowa HakkoDC92-B, ditrisarubicin B, Shionogi DOB-41, doxorubicin,doxorubicin-fibrinogen, elsamicin-A, epirubicin, erbstatin, esorubicin,esperamicin-A1, esperamicin-Alb, Erbamont FCE-21954, Fujisawa FK-973,fostriecin, Fujisawa FR-900482, glidobactin, gregatin-A, grincamycin,herbimycin, idarubicin, illudins, kazusamycin, kesarirhodins, KyowaHakko KM-5539, Kirin Brewery KRN-8602, Kyowa Hakko KT-5432, Kyowa HakkoKT-5594, Kyowa Hakko KT-6149, American Cyanamid LL-D49194, Meiji SeikaME 2303, menogaril, mitomycin, mitoxantrone, SmithKline M-TAG,neoenactin, Nippon Kayaku NK-313, Nippon Kayaku NKT-01, SRIInternational NSC-357704, oxalysine, oxaunomycin, peplomycin, pilatin,pirarubicin, porothramycin, pyrindamycin A, Tobishi RA-I, rapamycin,rhizoxin, rodorubicin, sibanomicin, siwenmycin, Sumitomo SM-5887, SnowBrand SN-706, Snow Brand SN-07, sorangicin-A, sparsomycin, SSPharmaceutical SS-21020, SS Pharmaceutical SS-7313B, SS PharmaceuticalSS-9816B, steffimycin B, Taiho 4181-2, talisomycin, Takeda TAN-868A,terpentecin, thrazine, tricrozarin A, Upjohn U-73975, Kyowa HakkoUCN-10028A, Fujisawa WF-3405, Yoshitomi Y-25024 and zorubicin.

A fourth family of anti-proliferative agents that may be used incombination therapy with the RRAmods consists of synthetic nucleosides.Several synthetic nucleosides have been identified that exhibitanticancer activity. A well known nucleoside derivative with stronganticancer activity is 5-fluorouracil (5-FU). 5-Fluorouracil has beenused clinically in the treatment of malignant tumors, including, forexample, carcinomas, sarcomas, skin cancer, cancer of the digestiveorgans, and breast cancer. 5-Fluorouracil, however, causes seriousadverse reactions such as nausea, alopecia, diarrhea, stomatitis,leukocytic thrombocytopenia, anorexia, pigmentation, and edema.Derivatives of 5-fluorouracil with anti-cancer activity have beendescribed in U.S. Pat. No. 4,336,381, which is herein incorporated byreference in its entirety. Further 5-FU derivatives have been describedin the following patents listed in JP 50-50383, JP 50-50384, JP50-64281, JP 51-146482, and JP 53-84981 hereby individually incorporatedby reference herein. Further synthetic nucleoside analogs include4-amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-one(e.g., 5-aza-21-deoxycytidine, decitabine, or DACOGEN, Eisai Inc.,Woodcliff Lake, N.J.). Other examples, of nucleoside analogs that can beused to treat cancer are listed in U.S. Pat. No. 4,000,137, which isincorporated herein by reference, Cytosine arabinoside (also referred toas Cytarabin, araC, and Cytosar) and 5-Azacytidine (VIDAZA, CelegeneCorp., Summit, N.J.).

A fifth family of anti-proliferative agents that may be used incombination therapy with the RRAmods consists of hormonal agents.Examples of hormonal-type anti-proliferative agents that may be usedinclude, but are not limited to Abarelix; Abbott A-84861; Abirateroneacetate; Aminoglutethimide; anastrozole; Asta Medica AN-207; Antide;Chugai AG-041R; Avorelin; aseranox; Sensus B2036-PEG; Bicalutamide;buserelin; BTG CB-7598; BTG CB-7630; Casodex; cetrolix; clastroban;clodronate disodium; Cosudex; Rotta Research CR-1505; cytadren; crinone;deslorelin; droloxifene; dutasteride; Elimina; Laval University EM-800;Laval University EM-652; epitiostanol; epristeride; Mediolanum EP-23904;EntreMed 2-ME; exemestane; fadrozole; finasteride; flutamide;formestane; Pharmacia & Upjohn FCE-24304; ganirelix; goserelin; Shiregonadorelin agonist; Glaxo Wellcome GW-5638; Hoechst Marion RousselHoe-766; NCI hCG; idoxifene; isocordoin; Zeneca ICI-182780; ZenecaICI-118630; Tulane University J015X; Schering Ag J96; ketanserin;lanreotide; Milkhaus LDI-200; letrozol; leuprolide; leuprorelin;liarozole; lisuride hydrogen maleate; loxiglumide; mepitiostane;Leuprorelin; Ligand Pharmaceuticals LG-1127; LG-1447; LG-2293; LG-2527;LG-2716; Bone Care International LR-103; Lilly LY-326315; LillyLY-353381-HCl; Lilly LY-326391; Lilly LY-353381; Lilly LY-357489;miproxifene phosphate; Orion Pharma MPV-2213ad; Tulane UniversityMZ-4-71; nafarelin; nilutamide; Snow Brand NKS01; octreotide; Azko NobelORG-31710; Azko Nobel ORG-31806; orimeten; orimetene; orimetine;ormeloxifene; osaterone; Smithkline Beecham SKB-105657; Tokyo UniversityOSW-1; Peptech PTL-03001; Pharmacia & Upjohn PNU-156765; quinagolide;ramorelix; Raloxifene; statin; sandostatin LAR; Shionogi S-10364;Novartis SMT-487; somavert; somatostatin; tamoxifen; tamoxifenmethiodide; teverelix; toremifene; triptorelin; TT-232; vapreotide;vorozole; Yamanouchi YM-116; Yamanouchi YM-511; Yamanouchi YM-55208;Yamanouchi YM-53789; Schering AG ZK-1911703; Schering AG ZK-230211; andZeneca ZD-182780.

A sixth family of anti-proliferative agents that may be used incombination therapy with the RRAmods consists of a miscellaneous familyof antineoplastic agents including, but not limited to alpha-carotene,alpha-difluoromethyl-arginine, acitretin, Biotec AD-5, Kyorin AHC-52,alstonine, amonafide, amphethinile, amsacrine, Angiostat, ankinomycin,anti-neoplaston A10, antineoplaston A2, antineoplaston A3,antineoplaston A5, antineoplaston AS2-1, Henkel APD, aphidicolinglycinate, asparaginase, Avarol, baccharin, batracylin, benfluron,benzotript, Ipsen-Beaufour BIM-23015, bisantrene, Bristo-MyersBMY-40481, Vestar boron-10, bromofosfamide, Wellcome BW 502, WellcomeBW-773, calcium carbonate, Calcet, Calci-Chew, Calci-Mix, Roxane calciumcarbonate tablets, caracemide, carmethizole hydrochloride, AjinomotoCDAF, chlorsulfaquinoxalone, Chemes CHX-2053, Chemex CHX-100,Warner-Lambert CI-921, Warner-Lambert CI-937, Warner-Lambert CI 941,Warner-Lambert CI-958, clanfenur, claviridenone, ICN compound 1259, ICNcompound 4711, Contracan, Cell Pathways CP-461, Yakult Honsha CPT-11,crisnatol, curaderm, cytochalasin B, cytarabine, cytocytin, Merz D-609,DABIS maleate, dacarbazine, datelliptinium, DFMO, didemnin-B,dihaematoporphyrin ether, dihydrolenperone, dinaline, distamycin, ToyoPharmar DM-341, Toyo Pharmar DM-75, Daiichi Seiyaku DN-9693, docetaxel,Encore Pharmaceuticals E7869, elliprabin, elliptinium acetate, TsumuraEPMTC, ergotamine, etoposide, etretinate, Eulexin®, Cell PathwaysExisulind® (sulindac sulphone or CP-246), fenretinide, Merck ResearchLabs Finasteride, Florical, Fujisawa FR-57704, gallium nitrate,gemcitabine, genkwadaphnin, Gerimed, Chugai GLA-43, Glaxo GR 63178,grifolan NMF-5N, hexadecylphosphocholine, Green Cross HO-221,homoharringtonine, hydroxyurea, BTG ICRF-187, ilmofosine, irinotecan,isoglutamine, isotretinoin, Otsuka JI-36, Ramot K 477, ketoconazole,Otsuak K-76COONa, Kureha Chemical K-AM, MECT Corp KI-8110, AmericanCyanamid L-623, leucovorin, levamisole, leukoregulin, lonidamine,Lundbeck LU-23-112, Lilly LY 186641, Materna, NCI (US) MAP, marycin,Merrel Dow MDL-27048, Medco MEDR-340, megestrol, merbarone, merocyaninederivatives, methylanilinoacridine, Molecular Genetics MGI-136,minactivin, mitonafide, mitoquidone, Monocal, mopidamol, motretinide,Zenyaku Kogyo MST-16, Mylanta, N (retinoyl)amino acids, Nilandron;Nisshin Flour Milling N-021, N-acylated-dehydroalanines, nafazatrom,Taisho NCU-190, Nephro-Calci tablets, nocodazole derivative, Normosang,NCI NSC-145813, NCI NSC-361456, NCI NSC-604782, NCI NSC-95580,octreotide, Ono ONO-112, oquizanocine, Akzo Org 10172, paclitaxel,pancratistatin, pazelliptine, Warner-Lambert PD-111707, Warner-LambertPD-115934, Warner-Lambert PD-131141, Pierre Fabre PE-1001, ICRT peptideD, piroxantrone, polyhaematoporphyrin, polypreic acid, Efamol porphyrin,probimane, procarbazine, proglumide, Invitron protease nexin I, TobishiRA-700, razoxane, retinoids, Encore Pharmaceuticals R-flurbiprofen,Sandostatin; Sapporo Breweries RBS, restrictin-P, retelliptine, retinoicacid, Rhone-Poulenc RP-49532, Rhone-Poulenc RP-56976, Scherring-PloughSC-57050, Scherring-Plough SC-57068, seienium (selenite andselenomethionine), SmithKline SK&F-104864, Sumitomo SM-108, KuraraySMANCS, SeaPharm SP-10094, spatol, spirocyclopropane derivatives,spirogermanium, Unimed, SS Pharmaceutical SS-554, strypoldinone,Stypoldione, Suntory SUN 0237, Suntory SUN 2071, Sugen SU-101, SugenSU-5416, Sugen SU-6668, sulindac, sulindac sulfone; superoxidedismutase, Toyama T-506, Toyama T-680, taxol, Teijin TEI-0303,teniposide, thaliblastine, Eastman Kodak TJB-29, tocotrienol, Topostin,Teijin TT-82, Kyowa Hakko UCN-01, Kyowa Hakko UCN-1028, ukrain, EastmanKodak USB-006, vinblastine, vinblastine sulfate, vincristine, vindesine,vinestramide, vinorelbine, vintriptol, vinzolidine, withanolides,Yamanouchi YM-534, Zileuton, ursodeoxycholic acid, and Zanosar.

In the instances of combination therapies described herein, it will beunderstood the administration further includes a pharmaceutically ortherapeutically effective amount of the additional therapeutic agent inquestion. The second or additional therapeutic agents described hereinmay be administered in the doses and regimens known in the art or may beadministered in low doses.

In some embodiments, the administration of a RRAmod and an additionaltherapeutic agent can result in a synergistic effect. A “synergisticeffect” as used herein means the combined effect of two or moretherapeutic agents can be greater than the sum of the separate effectsof the agents alone. For example, the combined effect of a RRAmod, andan anticancer agent, such as metformin, can be greater than the sum ofthe separate effects of an RRAmod and metformin alone.

Where the combined effect of administering a RRAmod and anothertherapeutic agent is greater than the sum of the separate effects of theRRAmod and the other agent alone, the RRAmod and/or therapeutic agentcan be administered to the subject in a lower dose or even asub-therapeutic dose. A benefit of lowering the dose of the combinationtherapeutic agents and therapies can include a decrease in the incidenceof adverse effects associated with higher dosages. For example, by thelowering the dosage of a chemotherapeutic agent such as methotrexate, areduction in the frequency and the severity of nausea and vomiting willresult when compared to that observed at higher dosages.

The additional therapeutic agent can be administered by a route and inan amount commonly used therefore, contemporaneously or sequentiallywith a RRAmod compound. When administered as a combination, a RRAmodcompound and additional therapeutic agent(s) can be formulated asseparate compositions which are given at the same time or differenttimes, or the therapeutic agents can be given as a single composition.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention

Example 1 Allosteric Modulators of Ribonucleotide Reductase (RR1)Hexamer Interface of RR1

Until now, the modulation of RR activity by small molecules binding atthe hexamer interface was unknown. Using X-ray Crystallography, we havemapped out a new druggable site of ribonucleotide reductase bound bypotent protein inhibitor small molecules that modulates its activity. Wehave discovered the epitope targeted by allosteric effectors on thelarge subunit of ribonucleotide reductase. The epitope includes residues1 to 21 of the N terminus of ribonucleotide reductase I bearing thesequence MHVIKRDGRQERVMFDKITSR (SEQ ID NO:1). However, the sequence alsoincludes residues 1 to 30.

Our discovery shows that small molecules will bind this region andallosterically modulate the enzyme. Moreover, small molecules inhibitthe ribonucleotide reductase hexamer formation.

Screening for RR Allosteric Inhibitors (RRAmods)

Using the Cincinnati chemical library and fluorescence-based assaysadapted for HTS we conducted a high throughput screen (HTS) for smallmolecules that will bind at the hexamer interface of human RR1 andmodulate ribonucleotide reductase enzyme activity.

In Silico Virtual Screening

We conducted an in silico virtual screening of the Cincinnati libraryusing the drug discovery software SYBYLX1.3. Here, the library wassubject to docking of ligands using the SUFFLEX dock option in SYBYL.The best hits were discriminated using two scoring functions called, adocking score and the C-score. The docking score is theoreticallyequivalent to the negative logarithm of K_(d), while C-score is aconsensus scoring function. Hence, docking scores that equal to 6 wouldmean a theoretical K_(d) of micromolar. The maximum C-score that can beobtained is five. Based on these criteria, after virtually screening thelibrary, the best 250 hits were chosen. The template used for dockingwas the hexamer interface of RR1 reported by us in Fairman, et al. 2011,Nat. Struc. Mol. Biol. (see FIG. 1).

Tryptophan Fluorescence Quenching Assay

We established fluorescence-based assays where fluorescence quenchingoccurs upon small molecules binding at the hexamer interface of RR1. Wescreened the top 100 hits from the virtual screen using a trytophanfluorescence-based quenching assay.

Methods

We recorded the tryptophan fluorescence spectra of Hur1 (Humanribonucleotide reductase) and Hur1 in the presence of Drug 4. Thespectra were recorded using a Jobin-Yvon-Spex fluorescencespectrophotometer by exciting the sample at a wavelength of 295 nm. Thesamples were titrated with 65 μM of Drug 4 at RT. Extent of quenchingwas determined after correcting for the compound fluorescence in thesame buffer.

Results

Of the first 10 compounds to be tested, we have obtained 11 hits. Someof these candidate compounds achieved as much as 40-50% fluorescencequenching (See FIG. 12). We also observed that some of the compoundsinduce a blueshift in the wavelength indicating the promotion ofhydrophobic interactions leading to a possible tightening of thestructure.

DNA Synthesis Assay

In order to determine the relative activity of candidate drugs and anestimate of the general range where the IC50s would fall, we exposedcandidate compounds for 2 continuous days. The results are summarized inTable 1 below:

TABLE 1 DRUG ID ACTIVITY IC₅₀ Drug 1 most potent IC₅₀ ~1 uM or less Drug2 2^(nd) most potent IC₅₀ ~1-5 μm Drug 3 little or no activity at 100 μMnull Drug 4 a bit of activity IC₅₀ near 50-100 μm

MTT Assay I

We conducted an MTT assay to measure cell metabolism as a surrogate forcell number using A549, Non-Small Cell Lung cells; 231, Triple negativebreast cells; and LN229. All values are relative to untreated controls,corrected for any MTT absorbance due to media alone.

Method

The cells were treated with continuous doses of the indicated drugs forthree days, at which time the MTT assay was run. The control is listedas a dose of 0.1 uM because we plot on a log scale and wish to avoidtaking the log of 0 in the calculations.

The results are shown in FIGS. 2-8 and summarized below in Table 2.

TABLE 2 DRUG ID ACTIVITY IC₅₀ Drug 1 Steady effect on all three celllines IC₅₀s in the 0.8-2.0 μM range Drug 2 Shows an extremely dramaticeffect. IC₅₀ between Visually the cells have undergone 3-12 uM eithernecrosis or apoptosis, and few intact cells are evident once they hittheir respective toxic dose Drug 3 virtually without effect, even at 100null μM Drug 4 some toxicity at 100 μM, but little null or none at 50 uM

MTT Assay II

We conducted an MTT assay to measure cell metabolism as a surrogate forcell number using A549, Non-Small Cell Lung cells; pancreatic cells(Panc-1); and colon cells (HCT-116). The results for Drugs I-III areshown in FIGS. 9-11 and summarized in Table 3 below. In additionGemcitabien IC₅₀s were found to be between 100 nm-500 nm.

TABLE 3 Docking Drug # Batch GRI Compound Weight Score IC₅₀ (μM) 1140247 253941

538 8.27 0.6-2   2 220435 193840

722 7.52 3-8 4 528949 258256

460 7.2  30-70

Example 2

We conducted a high throughput screen using the Cincinnati drug library(the former Proctor and Gamble drug library). We used amultidisciplinary screening approach as follows. (1) We used in silicodocking experiments with the N-terminus of hRRM1 to discover smallmolecule ligands (2) We tested the highest-ranking hits for hRRM1binding using a fluorescence quenching assay (FIG. 13) (3) We tested theligands that bound to hRRM1 in cell culture experiments to assess theirability to kill cancer cell lines (FIGS. 14 and 15), (4) the ligandsthat demonstrated anticancer properties for RR inhibition (Table 4) and(5). Finally, we attempted to co-crystallize RR with the ligands or soakthem into hRRM1 crystals to obtain their crystal structures. FIGS. 16Aand 16B shows the success of one such experiment, where we observed theligand to bind at the M-site, a site separate from the allosteric sites,the C site and the P site of hRRM1. This novel ligand-binding siteestablishes that we have discovered a new class of modulators of RRactivity. During step 3, several cell culture experiments were conductedto assess the potency of the RR modulators in their ability to killcancer. In one set of experiments, the modulators were tested asmonotherapies. One of the ligands had a concentration at half-maximalinhibition IC50 of approximately 200 nM, meaning it had a higher potencythan gemcitabine against colon and breast cancer cell lines (FIG. 15).Moreover, in a parallel study a modulator acted synergistically withgemcitabine improving gemcitabine's therapeutic index (FIG. 15). Theseexciting initial results may pave the way to develop a new class ofsafer anticancer agents. In this project we propose to study themechanism of action of this new class of RR modulators using astructural and biochemical approach.

TABLE 4 Compound Number Compound Structure IC₅₀ (μM) Compound 1

0.6-2   Compound 2

3-8 Compound 3

0.225 Compound 4

 5-10 Compound 5

 5-10 Compound 6

30

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications Such improvements,changes and modifications are within the skill of the art and areintended to be covered by the appended claims. All publications,patents, and patent applications cited in the present application areherein incorporated by reference in their entirety.

Having described the invention, the following is claimed:
 1. A method ofmodulating ribonucleotide reductase activity in a neoplastic cellcomprising administering to the cell an amount of a ribonucleotidereductase allosteric modulator (RRAmod), the amount being the amounteffective to inhibit neoplastic cell growth.
 2. The method of claim 1,the RRAmod modulating ribonucleotide reductase activity by selectivelybinding at the hexamer interface of RR1.
 3. The method of claim 2, thehexamer interface of RR1 comprising an epitope having an amino acidsequence corresponding to SEQ ID NO:
 1. 4. The method of claim 1,wherein modulating ribonucleotide reductase activity comprisesmodulating ribonucleotide reductase mediated catalyzation ofribonucleotides to deoxy ribonucleotides in the neoplastic cell, therebyunbalancing the nucleotide pool of DNA precursor molecules required forde novo DNA synthesis.
 5. The method of claim 1, the RRAmod comprising asmall molecule, a peptide, a peptidomimetic, or an antibody.
 6. Themethod of claim 1, the RRAmod comprising a small molecule having theformula:

wherein each of R₁ to R₂₀, R₄₆ to R₆₃ can be independently selected fromthe group consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl,hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄alkynyloxy, C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl(—CO-alkyl) and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl),C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl(—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO⁻),carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH₂), carbamide(—NH—(CO)—NH₂), cyano(—CN), isocyano (—N⁺C⁻), cyanato (—O—CN),isocyanato (—O—N⁺═C⁻), isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl(—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino,C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido(—NH—(CO)-aryl), imino (—CR═NH where R is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino (—CR═N(alkyl),where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂),combinations thereof, and wherein adjacent R groups may be linked toform a cyclic or polycyclic ring, wherein the cyclic ring is aromatic,alicyclic, heteroaromatic, or heterocyclic; X₁ is a C₁-C₂₄ alkyl, C₂-C₂₄alkenyl, C₂-C₂₄ alkynyl; Y₁ is a C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl; R₄₅ is hydrogen, a C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, an amino (—NH₂), or —(C₁-C₂₄ alkyl)-amino, and combinationsthereof; and a pharmaceutically acceptable salt thereof.
 7. The methodof claim 6, wherein the RRAmod is a small molecule selected from thegroup consisting of:

and pharmaceutically acceptable salts thereof.
 8. The method of claim 1,the cell comprising a cancer cell.
 9. The method of claim 8, the cancercell comprising a pancreatic, breast, lung, colon or glyoblastoma cancercell.
 10. A method of treating a neoplastic disorder in a subjectcomprising: administering to neoplastic cells of the subject atherapeutically effective amount of a pharmaceutical composition, thecomposition comprising a ribonucleotide reductase allosteric modulator(RRAmod), the therapeutically effective amount being the amount toinhibit neoplastic cell growth in the subject.
 11. The method of claim10, the RRAmod modulating ribonucleotide reductase activity byselectively binding to the hexamer interface of RR1.
 12. The method ofclaim 11, the hexamer interface of RR1 comprising an epitope having anamino acid sequence corresponding to SEQ ID NO:
 1. 13. The method ofclaim 10, wherein modulating ribonucleotide reductase activity comprisesmodulating ribonucleotide reductase mediated catalyzation ofribonucleotides to deoxy ribonucleotides in the cell, therebyunbalancing the nucleotide pool of DNA precursor molecules required forde novo DNA synthesis.
 14. The method of claim 10, the RRAmod comprisinga small molecule having the formula:

wherein each of R₁ to R₂₀, R₄₆ to R₆₃ can be independently selected fromthe group consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl,hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄alkynyloxy, C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl(—CO-alkyl) and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl),C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl(—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO⁻),carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH₂), carbamide(—NH—(CO)—NH₂), cyano(—CN), isocyano (—N⁺C⁻), cyanato (—O—CN),isocyanato (—O—N⁺═C⁻), isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl(—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino,C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido(—NH—(CO)-aryl), imino (—CR═NH where R is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino (—CR═N(alkyl),where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂),combinations thereof, and wherein adjacent R groups may be linked toform a cyclic or polycyclic ring, wherein the cyclic ring is aromatic,alicyclic, heteroaromatic, or heterocyclic; X₁ is a C₁-C₂₄ alkyl, C₂-C₂₄alkenyl, C₂-C₂₄ alkynyl; Y₁ is a C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl; R₄₅ is hydrogen, a C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, an amino (—NH₂), or —(C₁-C₂₄ alkyl)-amino, and combinationsthereof; and a pharmaceutically acceptable salt thereof.
 15. The methodof claim 14, wherein the RRAmod is a small molecule selected from thegroup consisting of:

and pharmaceutically acceptable salts thereof.
 16. The method of claim10, the neoplastic disorder comprising cancer.
 17. The method of claim16, wherein the cancer includes pancreatic, breast, lung, colon orglyoblastoma cancer.
 18. The method of claim 10, further administeringanother therapeutic agent in conjunction with the RRAmod.
 19. The methodof claim 18, the other therapeutic agent comprising at least one of achemotherapeutic agent, an antimetabolite, a DNA damaging agent, aribonucleotide reductase inhibiting agent, an antitumorgenic agent, anantimitotic agent, an antiviral agent, an antineoplastic agent, animmunotherapeutic agent, and a radiotherapeutic agent.
 20. Apharmaceutical composition comprising a ribonucleotide reductaseallosteric modulator (RRAmod), the RRAmod inhibiting cell growth whenadministered to a neoplastic cell, wherein the RRAmod includes a smallmolecule having the formula:

wherein each of R₁ to R₂₀, R₄₆ to R₆₃ can be independently selected fromthe group consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, C₃-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl,hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄alkynyloxy, C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl(—CO-alkyl) and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl),C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl(—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO⁻),carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH₂), carbamide(—NH—(CO)—NH₂), cyano(—CN), isocyano (—N⁺C⁻), cyanato (—O—CN),isocyanato (—O—N⁺═C⁻), isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl(—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino,C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido(—NH—(CO)-aryl), imino (—CR═NH where R is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino (—CR═N(alkyl),where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂),combinations thereof, and wherein adjacent R groups may be linked toform a cyclic or polycyclic ring, wherein the cyclic ring is aromatic,alicyclic, heteroaromatic, or heterocyclic; X₁ is a C₁-C₂₄ alkyl, C₂-C₂₄alkenyl, C₂-C₂₄ alkynyl; Y₁ is a C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl; R₄₅ is hydrogen, a C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, an amino (—NH₂), —(C₁-C₂₄ alkyl)-amino, and combinationsthereof; and a pharmaceutically acceptable salt thereof.
 21. Thepharmaceutical composition of claim 20, wherein the RRAmod is a smallmolecule selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 22. The pharmaceuticalcomposition of claim 20, the neoplastic cell comprising a cancer cell.23. The pharmaceutical composition of claim 24, the cancer cellcomprising a pancreatic, breast, lung, colon or glyoblastoma cancercell.
 24. The pharmaceutical composition of claim 20, further comprisinganother therapeutic agent in conjunction with the RRAmod.
 25. Thepharmaceutical composition of claim 24, the other therapeutic agentcomprising at least one of a chemotherapeutic agent, an antimetabolite,a DNA damaging agent, a ribonucleotide reductase inhibiting agent, anantitumorgenic agent, an antimitotic agent, an antiviral agent, anantineoplastic agent, an immunotherapeutic agent, and a radiotherapeuticagent.